The capacity of Staphylococcus aureus to express resistance to multiple antibiotics is a major global health problem. The recent emergence of strains with decreased susceptibility to vancomycin (VISA isolates) is particularly worrisome. New and effective antimicrobials against S. aureus are therefore needed. Recent attention has been paid to antimicrobial peptides (APs) as possible therapeutic agents in combating the problem of antibiotic-resistant strains of bacteria. Our research program and the studies of others have implicated human lysosomal cathepsin G (cat G) as a potent mediator of neutrophil killing of S. aureus. We have discovered an AP within the full-length cat G sequence that has potent in vitro activity against S. aureus (including VISA isolates and methicillin-resistant strains); this peptide comprises residues 117-136 of cat G and is termed CG 117-136. During the past funding period we successfully modified CG 117-136 to enhance its bactericidal activity. We also found that levels of staphylococcal susceptibility to CG 117-136 were linked to the major cold shock gone cspA. Loss of cspA expression due to Tn551insertional mutagenesis or deletion resulted in decreased susceptibility of S. aureus to this peptide. These cspA mutations also resulted in decreased or increased expression of several proteins and loss of pigmentation production. Moreover, growth of S. aureus in the presence of a sublethal concentration of this AP impacted the expression of several genes. In this proposal we will use a combination of microbial genetic (mutant selection), biochemical (proteomic analysis) and molecular (microarray gene chip technology) techniques to extend these observations with the goal of defining how S. aureus responds to AP. We will first define the role of cold shock gene expression in determining levels of AP susceptibility and pigment production (Specific Aim 1). The mechanisms by which cold shock proteins (CSPs) regulate S. aureus genes will be determined (Specific Aim 2). As exposure to AP represents a stress situation for bacteria, we will examine the genomic response of S. aureus to CG 117-136 by determining the genes of S. aureus that are regulated at the level of transcription during exposure to APs (Specific Aim 3). The integration of the genetic, molecular and biochemical experiments proposed will advance our knowledge regarding the mechanism of AP-killing of S. aureus and how this pathogen may develop resistance to this activity. This information should help in the development of new effective peptide-based drugs that could be used in the future to combat staphylococcal diseases caused by antibiotic-resistant strains.